Search PubMed⌕ Search

Biomedical subjects

Francisco E Baralle

Publications and source records attributed to Francisco E Baralle.

At least 19 recordsLinked to original sources
PubMed2006

Reduced splicing efficiency induced by synonymous substitutions may generate a substrate for natural selection of new splicing isoforms: the case of CFTR exon 12.

Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms.

Alternative Splicing↗
PubMed2006

Complex splicing control of the human Thrombopoietin gene by intronic G runs.

The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1-4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3' splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3-5 guanosines (namely, G1-G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7-G10 work in a combinatorial way to control the selection of the proper 3' splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3' splice site so providing functional evidence that this factor is involved in the selection of the authentic 3' splice site of THPO IVS2.

Alternative Splicing↗
PubMed2006

Defective splicing, disease and therapy: searching for master checkpoints in exon definition.

The number of aberrant splicing processes causing human disease is growing exponentially and many recent studies have uncovered some aspects of the unexpectedly complex network of interactions involved in these dysfunctions. As a consequence, our knowledge of the various cis- and trans-acting factors playing a role on both normal and aberrant splicing pathways has been enhanced greatly. However, the resulting information explosion has also uncovered the fact that many splicing systems are not easy to model. In fact we are still unable, with certainty, to predict the outcome of a given genomic variation. Nonetheless, in the midst of all this complexity some hard won lessons have been learned and in this survey we will focus on the importance of the wide sequence context when trying to understand why apparently similar mutations can give rise to different effects. The examples discussed in this summary will highlight the fine 'balance of power' that is often present between all the various regulatory elements that define exon boundaries. In the final part, we shall then discuss possible therapeutic targets and strategies to rescue genetic defects of complex splicing systems.

Exons↗
PubMed2006

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.

Alternative Splicing↗
PubMed2006

TDP43 depletion rescues aberrant CFTR exon 9 skipping.

CFTR exon 9 presents a 3' splice site polymorphism, (UG)mU(n), whose composition influences splicing. TDP43 specifically binds the UG tract of the transcript and inhibits splicing in vitro. We report that depletion of TDP43 through RNA interference removes splicing inhibition caused by unfavorable (UG)mU(n) sequences, indicating that TDP43 exerts a potent inhibitory effect in vivo. We also show that the UG-TDP43 interaction has a dominant role over other exon 9 splicing regulatory elements. These results suggest that TDP43 association near a splice site has determined the evolution of positive splicing regulatory elements to contrast this inhibition.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2006

Brain-specific promoter and polyadenylation sites of the beta-adducin pre-mRNA generate an unusually long 3'-UTR.

Adducins are a family of membrane skeleton proteins composed of alpha-, beta- and gamma-subunits that promote actin and spectrin association in erythrocytes. The alpha- and gamma-subunits are expressed ubiquitously, while the beta-subunit is found in brain and erythropoietic tissues. The brain beta-adducin protein is similar in size to that of spleen, but the mRNA transcript is a brain-specific one that has not been yet characterized, having an estimated length of 8-9 kb instead of the 3-4 kb of spleen mRNA. Here, we show the molecular basis for these differences by determining the structure of the brain-specific beta-adducin transcript in rats, mice and humans. We identified a brain-specific promoter in rodents that, apparently, was not conserved in humans. In addition, we present evidence that the brain-mRNAs are formed by a common mechanism consisting in the tissue-specific use of alternative polyadenylation sites generating unusually long 3'-untranslated region of up to 6.6 kb. This hypothesis is supported by the presence of highly-conserved regions flanking the brain-specific polyadenylation site that suggest the involvement of these sequences in the translational regulation, stability and/or subcellular localization of the beta-adducin transcript in the brain.

3' Untranslated Regions↗
PubMed2005

Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene.

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.

Apolipoprotein A-II↗
PubMed2005

TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing.

TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.

Alternative Splicing↗
PubMed2005

A polar mechanism coordinates different regions of alternative splicing within a single gene.

Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.

Alleles↗
PubMed2005

Functional studies on the ATM intronic splicing processing element.

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

Animals↗
PubMed2005

Human, Drosophila, and C.elegans TDP43: nucleic acid binding properties and splicing regulatory function.

TAR DNA binding protein (TDP43), a highly conserved heterogeneous nuclear ribonucleoprotein, was found to down-regulate splicing of the exon 9 cystic fibrosis transmembrane conductance regulator (CFTR) through specific binding to a UG-rich polymorphic region upstream of the 3' splice site. Despite the emergence of new information regarding the protein's nuclear localization and splicing regulatory activity, TDP43's role in cells remains elusive. To investigate the function of human TDP43 and its homologues, we cloned and characterized the proteins from Drosophila melanogaster and Caenorhabditis elegans. The proteins from human, fly, and worm show striking similarities in their nucleic acid binding specificity. We found that residues at two different positions, which show a strong conservation among TDP43 family members, are linked to the tight recognition of the target sequence. Our three-dimensional model of TDP43 in complex with a (UG)(m) sequence predicts that these residues make amino acid side-chain to base contacts. Moreover, our results suggest that Drosophila TDP43 is comparable to human TDP43 in regulating exon splicing. On the other hand, C.elegans TDP43 has no effect on exon recognition. TDP43 from C.elegans lacks the glycine-rich domain found at the carboxy terminus of the other two homologues. Mutants of human and fly TDP43 devoid of the C-terminal domain are likewise unable to affect splicing. Our studies suggest that the glycine-rich domain is essential for splicing regulation by human and fly TDP43.

Amino Acid Sequence↗
PubMed2005

Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution.

It is well established that exonic sequences contain regulatory elements of splicing that overlap with coding capacity. However, the conflict between ensuring splicing efficiency and preserving the coding capacity for an optimal protein during evolution has not been specifically analyzed. In fact, studies on genomic variability in fields as diverse as clinical genetics and molecular evolution mainly focus on the effect of mutations on protein function. Synonymous variations, in particular, are assumed to be functionally neutral both in clinical diagnosis and when measuring evolutionary distances between species. Using the cystic fibrosis transmembrane conductance regulator (CFTR) exon 12 splicing as a model, we have established that about one quarter of synonymous variations result in exon skipping and, hence, in an inactive CFTR protein. Furthermore, comparative splicing evaluation of mammalian sequence divergences showed that artificial combinations of CFTR exon 12 synonymous and nonsynonymous substitutions are incompatible with normal RNA processing. In particular, the combination of the mouse synonymous with the human missense variations causes exon skipping. It follows that there are two sequential levels at which evolutionary selection of genomic variants take place: splicing control and protein function optimization.

Amino Acid Sequence↗
PubMed2005

Dysregulation of the endothelial nitric oxide synthase-soluble guanylate cyclase pathway is normalized by insulin in the aorta of diabetic rat.

Antiatherogenic effects of nitric oxide (NO) are mediated by activation of soluble guanylate cyclase (sGC) and are impaired by diabetes in animals and humans. We investigated whether uncontrolled diabetes and insulin therapy effect expression and function of the main enzymes of the endothelial nitric oxide (eNOS)-sGC signaling pathway in vivo. Expression and function of eNOS, sGC and protein kinase G (PKG) were studied by Western blot analysis and vasorelaxation to NO-donor in thoracic aortas from control (CON) and streptozotocin (SZT)-induced diabetic rats during uncontrolled diabetes (DM) and insulin treatment (INS) for 8 weeks. Protein level of eNOS was increased (+300%, P < 0.05), while sGC (-50%) and PKG (-65%) proteins were reduced (P < 0.03) in aortas of DM. Insulin treatment normalized these defects resulting in eNOS, sGC and PKG aortic protein content comparable to control. In aortic rings, diethylamine NONOate (DEA-NONOate)-induced vasorelaxation was attenuated (P< or =0.05) in DM compared to control and returned to normal in INS. Thus, experimental diabetes decreases sGC and PKG expression and their NO-dependent activation in aorta despite overexpression of eNOS. These abnormalities are normalized by insulin treatment and improved metabolic control.

Animals↗
PubMed2005

Another step forward for SELEXive splicing.

Splicing alterations are being increasingly reported to cause human diseases. However, predicting beforehand whether a given mutation might lead to aberrant splicing is often hampered by insufficient knowledge of which cis-acting sequences affect exon recognition. To better define these sequences, experimental methods have been developed that integrate pre-mRNA splicing with sequence selection assays. Recently, a novel in vivo selection method based on a partially randomized full-length exon has enabled the identification of new splicing-controlling elements in SMN exon 7. Skipping of this exon results in an aberrant protein and is involved in proximal spinal muscular atrophy (SMA). Hopefully, this approach will provide novel targets for nascent RNA molecular medicine approaches for the recovery of abnormal pre-mRNA splicing events.

Alternative Splicing↗
PubMed2004

The erythrocyte skeletons of beta-adducin deficient mice have altered levels of tropomyosin, tropomodulin and EcapZ.

The erythrocyte membrane cytoskeleton is organized as a polygonal spectrin network linked to short actin filaments that are capped by adducin at the barbed ends. We have constructed a mouse strain deficient in beta-adducin having abnormal erythrocytes. We show here that the levels of several skeletal proteins from beta-adducin mutant erythrocytes are altered. In fact, CapZ, the main muscle actin-capping protein of the barbed ends that in the erythrocytes is cytoplasmic, is 9-fold upregulated in mutant skeletons of erythrocytes suggesting a compensatory mechanism. We also detected upregulation of tropomodulin and downregulation of alpha-tropomyosin and actin. In addition, purified adducin can be re-incorporated into adducin-deficient ghosts.

Actins↗
PubMed2004

An intronic mutation causes long QT syndrome.

OBJECTIVES: The purpose of this research was to determine whether an intronic variant (T1945+6C) in KCNH2 is a disease-causing mutation, and if expanded phenotyping criteria produce improved identification of long QT syndrome (LQTS) patients. BACKGROUND: Long QT syndrome is usually caused by mutations in conserved coding regions or invariant splice sites, yet no mutation is found in 30% to 50% of families. In one such family, we identified an intronic variant in KCNH2. Long QT syndrome diagnosis is hindered by reduced penetrance, as the long QT phenotype is absent on baseline electrocardiogram (ECG) in about 30%. METHODS: Fifty-two family members were phenotyped by baseline QTc, QTc maximum on serial ECGs (Ser QTc-max), and on exercise ECGs (Ex QTc-max) and by T-wave patterns. Linkage analysis tested association of the intronic change with phenotype. The consequences of T1945+6C on splicing was studied using a minigene system and on function by heterologous expression. RESULTS: Expanded phenotype/pedigree criteria identified 23 affected and 29 unaffected. Affected versus unaffected had baseline QTc 484 +/- 48 ms versus 422 +/- 20 ms, Ser QTc-max 508 +/- 48 ms versus 448 +/- 10 ms, Ex QTc-max 513 +/- 54 ms versus 444 +/- 11 ms, and LQT2 T waves in 87% versus 0%. Linkage analysis demonstrated a logarithm of odds score of 10.22. Splicing assay showed T1945+6C caused downstream intron retention. Complementary deoxyribonucleic acid with retained intron 7 failed to produce functional channels. CONCLUSIONS: T1945+6C is a disease-causing mutation. It alters KCNH2 splicing and cosegregates with the LQT2 phenotype. Expanded ECG criteria plus pedigree analysis provided accurate clinical diagnosis of all carriers including those with reduced penetrance. Intronic mutations may be responsible for LQTS in some families with otherwise negative mutation screening.

Adult↗